Antibiotic LL-E19020 gamma

ABSTRACT

The invention provides an antibiotic designated LL-E19020 Gamma which is derived from the microorganism Streptomyces lydicus ssp. tanzanius NRRL 18036.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a new antibiotic designated LL-E19020 Gamma, toits production by fermentation and to a process for its recovery andpurification.

2. Description of the Prior Art

Antibiotics LL-E19020 Alpha and LL-E19020 Beta are disclosed in U.S.Pat. No. 4,705,688, The Journal Of Antibiotics, 41(10), 1511-1514 (1988)and The Journal Of Antibiotics, 42(10), 1489-1493 (1989). AntibioticLL-E19020 Alpha has a phenylacetate ester group attached at C-23 and hasthe structure: ##STR1##

Antibiotic LL-E19020 Beta has a phenylacetate ester group attached atC-24 and has the structure: ##STR2##

A process for purification of the antibiotic LL-E19020 Alpha by reversedphase HPLC purification is described in Journal Of Chromatography, 484,381-390(1989). Antibiotics LL-E19020 Alpha and LL-E 19020 Beta are alsouseful for increasing the growth rate of meat producing animals and fortreating respiratory disease, fowl cholera and necrotic enteritis asdescribed in U.S. Pat. No. 4,704,276 and U.S. Pat. No. 4,968,493.

A related family of compounds, the phenelfamycins, is reported in TheJournal Of Antibiotics, 41(10), 1293-1299 (1988); The Journal OfAntibiotics, 41(10), 1300-1315 (1988); The Journal Of Antibiotics,39(10), 1361-1367 (1986); The Journal Of Antibiotics, 42(1), 94-101(1989); Antimicrobiol Agents and Chemotherapy, 33(3), 322-325 (1989);and Program and Abstracts Of The 27th Interscience Conference onAntimicrobial Agents Chemotherapy, No. 995, p 270, New York, Oct. 4-71987.

SUMMARY OF THE INVENTION

A new antibiotic designated LL-E19020 Gamma has now been found. Thestructure of the new antibiotic LL-E19020 Gamma is: ##STR3##

As can be determined from the above structure antibiotic LL-E19020 Gammadiffers from the previously known antibiotics LL-E19020 Alpha andLL-E19020 Beta in that LL-E19020 Gamma has a 4-hydroxyphenylacetateester group attached at C-23. The physico chemical characteristics ofLL-E19020 Gamma are as follows:

1. Molecular weight:1241 (FABMS=M/Z 1264 corresponding to M+Na!+)

2. Molecular formula:C₆₅ H₉₅ NO₂₂ microanalysisfound(calc):C62.22(62.85); H7.77(7.66); N 0.92(1.13). HRFABMS:(M+K)+=M/Z1280.5983 (calc. 1280.5983)

3. Specific optical rotation: α!_(D) 26° C.=-7° C. (1.001, MeOH)

4. Ultraviolet Absorption Spectrum as shown in Figure I. UV absorptionMeOH!λmax (ε):231 nm (58,600); 287 nm (39,300).

5. IR absorption spectrum as shown in Figure II. IR absorption spectrumKBr! ν max:3411br, 2974, 2934, 2828, 1735, 1716sh, 1700, 1652, 1647,1617, 1455, 1367, 1222, 1098, 1023 cm⁻¹.

6. Proton ¹ H NMR CDCl₃ !:Spectrum (300 MHz) as shown in Figure III.

7. Carbon 13 ¹³ C NMR CDCl₃ ! Spectrum as shown in Figure IV,significant peaks listed below (δ from TMS):

    ______________________________________                                        173.4     127.5          74.64  49.77                                         171.9     126.3          74.51  41.85                                         170.1     125.1          74.42  40.92                                         155.7     120.7          74.07  39.91                                         145.7     115.6(2×)                                                                              72.19  39.23                                         140.3     100.5          71.83  38.81                                         136.9     98.90          69.10  32.98                                         134.3     97.28          67.57  30.98                                         132.0     96.93          66.40  29.91                                         130.6(2×)                                                                         89.16          66.07  23.75                                         130.1     83.21          63.57  18.10                                         129.0     81.64          56.53  17.20                                         128.5     77.63          56.10  17.00                                         128.3     77.20          55.50  14.84                                         128.2     76.46          55.45  13.50                                         128.1     10.97          10.18                                                ______________________________________                                    

8. High pressure liquid chromatography (HPLC) retention time of 18.5minutes using a gradient of acetonitrile in aqueous acetic acid.

9. High pressure liquid chromatography (HPLC) retention time of 19.6minutes using a gradient of dioxane in aqueous acetic acid.

The new antibiotic LL-E19020 Gamma is formed along with LL-E19020 Alphaand LL-E19020 Beta during cultivation under controlled conditions of astrain of Streptomyces lydicus ssp. tanzanius, NRRL 18036. The newantibiotic LL-E19020 Gamma is separated from LL-E 19020 Alpha andLL-E19020 Beta and subsequently purified by high pressure liquidchromatography (HPLC).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ultraviolet absorption spectrum of LL-E19020 Gamma.

Figure 2 shows the infrared absorption spectrum of LL-E19020 Gamma.

Figure 3 shows the proton nuclear magnetic resonance spectrum ofLL-E19020 Gamma.

Figure 4 shows the carbon-13 nuclear magnetic resonance spectrum ofLL-E19020 Gamma.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The antibiotic LL-E19020 Gamma is produced by fermentation of a strainof Streptomyces lydicus, ssp. tanzanius, NRRL 18036, in an aqueousnutrient medium containing assimilable sources of carbon and nitrogenunder submerged aerobic conditions. This microorganism is maintained inthe culture collection of the Medical Research Division, AmericanCyanamid Company, Pearl River, N.Y. as culture number LL-E19020. Aviable culture of this new microorganism has been deposited with thePatent Culture Collection Laboratory, Northern Regional Research Center,U.S. Department of Agriculture, Peoria, Ill. 61604, and has been addedto its permanent collection. It has been assigned the strain designationNRRL 18036 by said depository.

Culture LL-E19020 produces short spiral spore chains, 10-50 spores long,with occasional longer chains. These tend to coalesce to form dryblackish masses on such ISP media as oatmeal and inorganic salts-starch.The spores have smooth surfaces as assessed by electron microscopy. Thestrain contains the L isomer of diaminopimelic acid, and may thus beassigned to the genus Streptomyces.

In the ISP tests for utilization of carbohydrates, LL-E19020 showsgrowth on arabinose, fructose, inositol, mannitol, reffinose, rhamnose,sucrose and xylose. Cellulose is not utilized.

The reactions of LL-E19020 in the Gordon physiological series arecompared in the following Table I with those of Streptomyces lydicus ISP5461 which it most closely resembles morphologically andphysiologically.

Because LL-E19020 differs from ISP 5461 in five(5) characteristics(xanthine hydrolysis, decarboxylation of oxalate, acid from erythritol,rhamnose and β-methyl-D-xyloside) it is designated as a subspecies ofStrentomyces lydicus.

                  TABLE I                                                         ______________________________________                                        Gordon Test Reactions Of LL-E19020                                            And Streptomyces lydicus ISP 5461                                             Reactions          LL-E19020 ISP 5461                                         ______________________________________                                        Degradation/Transformation of                                                 Casin              +         +                                                Xanthine           -         +                                                Hypoxanthine       +         +                                                Tyrosine           +         +                                                Adenine            +         +                                                Production of                                                                 Amylase            +         +                                                Gelatinase         +         +                                                Phosphatase        +         +                                                Nitrate Reductase  -         -                                                Urease             +         +                                                Esculinase         +         +                                                Growth on/in                                                                  5% Sodium chloride +         +                                                Salicylate         -         -                                                Lysozyme Broth     trace     trace                                            Utilization of                                                                Acetate            +         +                                                Benzoate           +         +                                                Citrate            +         +                                                Lactate            +         +                                                Malate             +         +                                                Mucate             +         +                                                Oxalate            +         -                                                Propionate         +         +                                                Pyruvate           +         +                                                Succinate          +         +                                                Tartrate           -         -                                                Growth at                                                                     10° C.      +         +                                                42° C.      -         -                                                50° C.      -         -                                                Acid from                                                                     Adonitol           +         +                                                Arbinose           +         +                                                Cellobiose         +         +                                                Dextrin            +         +                                                Dulcitol           -         -                                                Erythritol         +         -                                                Fructose           +         +                                                Galactose          +         +                                                Glucose            +         +                                                Glycerol           +         +                                                Inositol           +         +                                                Lactose            +         +                                                Maltose            +         +                                                Mannitol           +         +                                                Mannose            +         +                                                Melibiose          +         +                                                α-Methyl-D-Glucoside                                                                       +         +                                                Raffinose          +         +                                                Rhamnose           +         -                                                Salicin            +         +                                                Sobitol            +         +                                                Sucrose            +         +                                                Trehalose          +         +                                                Xylose             +         +                                                β-Methyl-D-Xyloside                                                                         +         -                                                ______________________________________                                    

It is to be understood that for the production of these newantibacterial agents the present invention is not limited to thisparticular organism or to organisms fully answering the abovecharacteristics which are given for illustrative purposes only. In fact,it is desired and intended to include the use of mutants produced fromthis organism by various means such as exposure of X-radiation,ultraviolet radiation, N'-methyl-N'-nitro-N-nitrosoguanidine,actinophages and the like.

Cultivation of Streptomyces lydicus SSP. tanzanius NRRL 18036 may becarried out in a wide variety of liquid culture media. Media which areuseful for the production of LL-E19020 Gamma include an assimilablesource of carbon, such as dextrin, sucrose, molasses, glycerol, etc; anassimilable source of nitrogen such as protein, protein hydrolysate,polypeptides, amino acids, corn steep liquor, etc; and inorganic anionsand cations, such as potassium, sodium, ammonium, calcium, sulfate,carbonate, phosphate, chloride, etc. Trace elements such as boron,molybdenum, copper, etc., are supplied as impurities of otherconstituents of the media. Aeration in tanks and bottles is supplied byforcing sterile air through or onto the surface of the fermentingmedium. Further agitation in tanks is provided by a mechanical impeller.An antifoam agent such as silicone oil may be added as needed.

The antibiotic LL-E19020 Gamma is recovered from the fermentation brothby extraction of the broth into a solvent such as ethyl acetate,followed by chromatography of the ethyl acetate extracted broth using ahigh pressure liquid chromatography with a twelve (12) liter reversephase column (C18 bonded phase 40 micron) using 0.1M ammonium acetate pH4.3/acetonitrile (1:1) to obtain a mixture of LL-E19020 Alpha andLL-E19020 Gamma. Additional purification of this mixture by highpressure liquid chromatography using a twelve (12) liter reverse-phasecolumn (C18 bonded phase 40 micron) using 0.1M ammonium acetate pH4.3/acetonitrile (1:1) gives pure LL-E19020 Gamma and pure LL-E19020Alpha.

EXAMPLE 1 Inoculum Preparation

A typical medium used to grow the primary inoculum is prepared accordingto the following formula:

    ______________________________________                                        Dextrose         1.0%                                                         Dextrin          2.0%                                                         Yeast extract    0.5%                                                         NZ Amine A       0.5%                                                         Calcium carbonate                                                                              0.1%                                                         Antifoam         0.3%                                                         Water qs         100.0%                                                       ______________________________________                                    

NOTE:NZ Amine A is a pancreatic digest of casein, registered trademarkof Scheffield Chemical, Norwich, N.Y.

This medium is sterilized and 100 ml, in a 500 ml flask, is inoculatedwith Streptomyces lydicusssp. tanzanius NRRL 18036. The medium is thenplaced on a rotary shaker and incubated at 28° C. for 48 hours providinga primary inoculum. This primary inoculum is the used to inoculate 10liters of the same sterile medium in a bottle. This culture is grown for24 hours providing secondary inoculum. This secondary inoculum is thenused to inoculate 300 liters of the same sterile medium in a tank. Thisculture is grown at 300° C. for 44 hours with a sterile air flow of 0.66liters per liter of mash per minute and agitation by an impeller drivenat 200 rpm, providing a tertiary inoculum.

EXAMPLE 2 Fermentation

A fermentation production medium of the following formulation isprepared:

    ______________________________________                                        Dextrin          7.0%                                                         Dextrose         0.5%                                                         Soy flour        1.5%                                                         Corn Steep liquor                                                                              0.5%                                                         Calcium carbonate                                                                              0.5%                                                         Silicone antifoam                                                                              0.3%                                                         Water qs         100.0%                                                       ______________________________________                                    

This medium is sterilized and 1500 liters is then inoculated with 150liters of tertiary inoculum from Example 1. The fermentation isconducted at 28° C. with a sterile air flow of 3.3 liters of air perliter of mash per minute and agitation by an impeller driven at 100 rpmfor 123 hours, at which time the mash is harvested.

EXAMPLE 3

A fermentation medium of the following formulation is prepared:

    ______________________________________                                        Dextrin          7.0%                                                         Dextrose         0.5%                                                         Soy flour        1.5%                                                         Corn Steep Liquor                                                                              0.5%                                                         Calcium carbonate                                                                              0.5%                                                         Silicone antifoam                                                                              0.3%                                                         Water qs         100.0%                                                       ______________________________________                                    

This medium is sterilized and 3000 liters is then inoculated with 300liters of tertiary inoculum similarly prepared as in Example 1. Thefermentation is conducted at 28° C. with a sterile air flow of 6.5liters of air per liter of mash per minute and agitation by an impellerdriven at 100 rpm for 89 hours, at which time the mash is harvested.

EXAMPLE 4 Isolation and Purification of LL-E19020 Gamma

The harvest mash from two (2) fermentations conducted as described inExample 2 and Example 3, making a total volume of 3200 liters, isdiluted with 1600 liters of methyl alcohol and filtered throughdiatomaceous earth. The filter cake is washed with 320 liters of waterand the wash is added to the filtrate giving a total volume of 5000liters. A 800 liter volume is charged to a still and evaporated to 500liters. This procedure is repeated until the total volume is reduced to2950 liters followed by dilution with 1450 liters of ethyl acetate. Thelower phase is removed and the upper phase of 900 liters evaporated to79.5 liters. This concentrate is diluted with 80 liters of ethyl acetateand the lower layer removed. The upper layer is evaporated to give 2.4liters of a syrup. This crude product is repeatedly decanted with hexanethen dissolved in methyl alcohol and applied portion wise to a 12 literreverse phase column (C18 bonded phase 40 micron). In a typical run, 400ml of syrup is dissolved in methyl alcohol to give a final volume of 700ml which is applied to the reverse phase column and eluted with 1:1 0.1Mammonium acetate:acetonitrile at pH 4.3 to afford upon evaporation ofthe volatiles 38 g of impure LL-E19020 Gamma. Several like runs arecompleted in this manner and the products combined.

Purification of LL-E19020 Alpha and LL-E19020 Gamma

A total of 100 g of impure LL-E19020 Gamma is charged to a 12 literreverse phase column (C18 bonded phase 40 micron) and eluted with 0.1Mammonium acetate buffer pH 4.3/acetonitrile (1:1). Fractions designatedF1-F28, each having a volume of 20 liters are collected. Fraction F4 isstirred with 15 liters of methylene chloride for 1 hour. The methylenechloride layer is separated and evaporated to 1 liter, dried withcalcium chloride and evaporated to a residue which is dissolved in 75 mlof methyl alcohol and filtered. The filtrate is added, 5 ml at a time,to a 2.2×25 cm(10 micron) reverse phase C18 chromatographic column. Thecolumn is eluted with 40% acetonitrile in 0.05M ammonium acetate buffer(pH 4.5) at a flow rate of 9.9 ml/minute. The eluate collected after 2.5to 3 hours is extracted with ethyl acetate. The organic layer isevaporated to a syrup which is dissolved in t-butanol and freeze driedto afford 160 mg of pure LL-E19020 Gamma.

ANALYTICAL HIGH PRESSURE LIOUID CHROMATOGRAPHY (HPLC)

The LL-E19020 Gamma component is analyzed using two different analyticalHPLC systems. Retention time compared to E19020 α and β are given in thetable below.

    ______________________________________                                                   RETENTION TIME (MINUTES)                                           COMPONENTS   SYSTEM A    SYSTEM B                                             ______________________________________                                        LL-E19020α                                                                           22.7        23.5                                                 LL-E19020β                                                                            27.6        26.7                                                 LL-E19020γ                                                                           18.5        19.6                                                 ______________________________________                                    

A. HPLC system:Alltech adsorbosphere HS 5μ C18 column (4.6×250mm) withguard column, eluted with a gradient of acetonitrile in 1% aqueousacetic acid. The starting composition is 40% acetonitrile linearlyincreasing to 70% over 25 minutes and holding at 70% for 5 minutes. Theflow rate is 1.0 mL per minute.

B. HPLC system:Alltech adsorbosphere HS 5μ C18 (4.6×250 mm) with guardcolumn, eluted with a gradient of dioxane in 1% aqueous acetic acid. Thestarting composition is 55% dioxane, increasing to 70% over 25 minutesand holding at 70% for 5 minutes. The flow rate is 1.0 mL per minutes.

EXAMPLE 5 In Vitro Antibacterial Activity Of LL-E19020 Gamma

The in vitro antibacterial activity of LL-E19020 Gamma is determinedagainst a spectrum of gram positive and gram negative bacteria by astandard agar dilution method. Mueller-Hinton agar containing 5% sheepblood and two-fold decreasing concentrations of LL-E19020 Gamma ispoured into petri dishes. The agar surfaces are inoculated with 1 to5×10⁴ colony forming units of bacteria by means of the Steersreplicating device. The lowest concentration of antibiotic that inhibitsgrowth of a bacterial strain after 18 hours incubation is recorded asthe minimal inhibitory concentration for that strain.

Minimum Inhibitory Concentration Procedure By Agar Dilution

1. Serial two-flow dilutions of drug are prepared in Mueller-Hintonbroth in a range of 2560 μg/ml-0.15 μg/ml plus a solvent control.

2. Two milliliters of drug dilution (10X) are added to sterile screw capbottles to which 18 ml of Mueller-Hinton agar containing 5.6%defibrinated sheep blood is added. Final drug concentration ranges 256μg/ml-0.015 μg/ml in agar containing 5% sheep blood.

3. A few isolated colonies of each test organism are inoculated into 5ml trypticase soy broth or brain heart infusion broth. The cultures areshaken at 35° C. for 5 hours.

4. Each culture is diluted 1:50 (10⁻¹.7) in Mueller-Hinton broth andapplied to agar plates using a Steers replicator. Control plates shouldbe seeded last to ensure that viable organisms are present throughoutthe procedure. Inoculated agar plates are allowed to stand undisturbeduntil the inoculum spots are completely absorbed.

5. The plates are inverted and incubated at 35° C. for 18 hours withCO₂.

6. The minimum inhibitory concentration (MIC) is taken as the lowestconcentration of antimicrobial agent at which complete inhibition ofantimicrobial agent at which complete inhibition occurs. A very fine,barely visible haze or a single colony is disregarded.

The results are as follows:

    ______________________________________                                        In Vitro Activity of LL-E19020 Gamma                                          MINIMAL INHIBITORY CONCENTRATION (MCG/ML)                                     ORGANISM               LL-E19020 GAMMA                                        ______________________________________                                        1.  Staphylococcus aureus NEMC 87-69                                                                     32                                                 2.  Staphylococcus aureus ROSE (MP)*                                                                     32                                                 3.  Staphylococcus aureus IVES 160                                                                       32                                                 4.  Staphylococcus aureus IVES 396                                                                       64                                                 5.  Staphylococcus aureus VGH 84-47                                                                      64                                                 6.  Staphylococcus aureus CMC 83-131                                                                     64                                                 7.  Staphylococcus aureus SMITH (MP)                                                                     128                                                8.  Staphylococcus aureus ATCC 25923                                                                     >128                                               9.  Staphylococcus aureus ATCC 29213                                                                     128                                                10. Staphylococcus haemolyticus AVAH 88-1                                                                64                                                 11. Staphylococcus haemolyticus AVAH 88-3                                                                16                                                 12. Staphylococcus eidermidis IVES 455                                                                   16                                                 13. Enterococcus spp. ARUM 87-41                                                                         8                                                  14. Enterococcus spp. CHBM 88-60                                                                         16                                                 15. Enterococcus spp. WRVA 88-33                                                                         16                                                 16. Enterococcus spp. UCI 85-30                                                                          16                                                 17. Enterococcus spp. VGH 84-69                                                                          16                                                 18. Enterococcus spp. CMC 83-120                                                                         16                                                 19. Streptococcus pyogenes AMCH 88-84                                                                    0.12                                               20. Streptococcus pyogenes AMCH 88-86                                                                    0.5                                                21. Streptococcus pyogenes C203 (MP)                                                                     0.12                                               22. Streptococcus pneumoniae SV-1 (MP)                                                                   0.12                                               23. Streptococcus pneumoniae CHBM 88-75                                                                  16                                                 24. Streptococcus pneumoniae TEX 85-2                                                                    0.5                                                25. Bacillus cereus DAVIES 32                                                 26. Klebsiella pneumoniae NEMC 87-271                                                                    >128                                               27. Escherichia coli ATCC 25922                                                                          >128                                               28. Escherichia coli ATCC 35218                                                                          >128                                               29. Pseudomonas aeruginosa 12-4-4 (MP)                                                                   >128                                               ______________________________________                                         *MP = Mouse passage used in in vivo studies                              

As can be seen from the in vitro data above, LL-E19020 Gammademonstrated no gram-negative activity (MIC >128 μg/ml), showed poor tomoderate activity vs staphylococci and enterococci (MIC 8-128 μg/ml) andrelatively good activity against non-enterococcal streptococci (MIC0.12-16 μg/ml).

EXAMPLE 6 In Vivo Activity Of LL-E19020 Gamma

The in vivo antibacterial activity of antibiotic LL-E19020 Gamma isestablished by infecting female CD-1 mice from Charles RiverLaboratories, weighing 20±2 g each, intraperitoneally with either 2.0×10 CFU/0.5 ml Streptococcus pyogenes C203 suspended in broth or 2.8×106CFU/0.5 ml Straphylococcus aureus Smith suspended in 5% hog gastricmucin. The mice were treated subcutaneously, 30 minutes after infectionwith the indicated dose of the test compound in 0.5 ml of 0.2% aqueousagar. Toxicity studies were performed administering the same treatmentto uninfected mice.

The results are as follows:

    ______________________________________                                        In Vivo Activity Of LL-E19020 Gamma                                           Survival Ratio 7 Days After Infection With                                    ______________________________________                                        Single Subcutaneous                                                                       S. pyogenes C203                                                                             S. aureus Smith                                    Dose (MG/KG)                                                                              LL-E19020 Gamma                                                                              LL-E19020 Gamma                                    ______________________________________                                        64          NT             0/5                                                32          NT             0/5                                                16          4/5            0/5                                                8           3/5            0/5                                                4           1/5            0/5                                                2           0/5            0/5                                                1           0/5            0/5                                                  0.5       0/5            0/5                                                ______________________________________                                        Single Subcutaneous                                                           Dose (MG/KG)   LL-E19020 Gamma                                                ______________________________________                                        64             2/2                                                            32             2/2                                                            16             2/2                                                            8              2/2                                                            4              2/2                                                            2              2/2                                                            1              2/2                                                              0.5          2/2                                                            ______________________________________                                    

The data above shows the in vivo activity of LL-E19020 gamma vs acutelethal infections in mice. LL-E19020 gamma had an approximate ED₅₀between 4-8 mg/kg/ssc vs Streptococcus pyogenes C203. Against theStaphylococcus aureus Smith infection LL-E19020 gamma had an approximateED₅₀ of >64 mg/kg/ssc. LL-E19020 gamma did not exhibit toxic symptomswhen dosed subcutaneously at the same levels used in the protectionstudy.

Antibiotic LL-E19020 Gamma derives its utility from antibacterialactivity. For example this antibiotic may be used in the suppression ofbacterial infections, as a topical antibacterial agent and as a generaldisinfectant for laboratories.

In addition to its antibacterial activity this compound is effective asan anticoccidial agent in poultry and as a growth promotant andanthelmintic agent. These utilities are the subject of patentapplications filed concurrently herewith and incorporated herein byreference.

In therapeutic use, the compound of this invention may be administeredin the form of a conventional pharmaceutical composition appropriate forthe intended use. Such a composition may be formulated so as to besuitable for oral, parenteral, or topical administration. The activeingredient may be combined in admixture with a nontoxic pharmaceuticallyacceptable carrier, which carrier may take a wide variety of formsdepending on the form of preparation desired for administration, ie,oral, parenteral or topical.

When the compound is employed for the above utility, it can be combinedwith one or more pharmaceutically acceptable carriers, for example,solvents, diluents and the like, and may be administered orally in suchforms as tablets, capsules, dispersible powders, granules, orsuspensions containing, for example, from about 0.05 to 5% of suspendingagent, syrups containing, for example from about 10 to 50% of sugar, andelixirs containing, for example, from about 20 to 50% ethanol, and thelike, or parenterally in the form of sterile injectable solutions orsuspensions containing from about 0.05 to 5% suspending agent in anisotonic medium. Such pharmaceutical preparations may contain, forexample, from about 0.05 up to about 90% of the active ingredient incombination with the carrier, more usually between about 5% and 60% byweight.

An effective amount of compound from 0.2 mg/kg of body weight to 100.0mg/kg of body weight should be administered one to five times per dayvia any topical route of administration including but not limited tooral, parenteral (including subcutaneous, intravenous, intramuscular,intrasternal injection or infusion techniques), by inhalation spray, orrectally, in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. It will beunderstood, however, that the specific dose level and frequency ofdosage for any particular patient may be varied and will depend upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the host undergoing therapy.

These active compounds may be administered orally as well as byintravenous, intramuscular, or subcutaneous routes. Solid carriersinclude starch, lactose, dicalcium phosphate, microcrystallinecellulose, sucrose and kaolin, while liquid carriers include sterilewater, polyethylene glycols, non-ionic surfactants and edible oils suchas corn, peanut and sesame oils, as are appropriate to the nature of theactive ingredient and the particular form of administration desired.Adjuvants customarily employed in the preparation of pharmaceuticalcompositions may be advantageously included, such as flavoring agents,coloring agents, preserving agents, and antioxidants, for example,vitamin E, ascorbic acid, BHT and BHA.

The preferred pharmaceutical compositions from the stand-point of easeof preparation and administration are solid compositions, particularlytablets and hard-filled or liquid-filled capsules. Oral administrationof the compound is preferred.

These active compounds may also be administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt can be prepared inwater suitably mixed with a surfactant such as hydroxy propylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oil.

We claim:
 1. A compound LL-E19020 Gamma comprising(a) the structure##STR4## (b) an elemental analysis:C 62.22; H 7.77; N 0.92; (c) amolecular weight of 1241 (FABMS=M/Z 1264 corresponding to M+Na!+); (d) aspecific optical rotation: α!_(D) 26° C.=-7° C.(1.001, MeOH) (e) acharacteristic ultraviolet absorption spectrum as shown in FIG. I of theattached drawings; (f) a characteristic infrared absorption spectrum asshown in Figure II of the attached drawings; (g) a characteristic protonnuclear magnetic resonance spectrum as shown in Figure III of theattached drawings; (h) a characteristic carbon-13 nuclear magneticresonance spectrum as shown in Figure IV of the attached drawings, (i) acharacteristic HPLC retention time of 18.5 minutes using a gradient ofacetonitrile in aqueous acetic acid; and (j) a characteristic HPLCretention time of 19.6 minutes using a gradient of dioxane in aqueousacetic acid.
 2. A method of treating bacterial infections in warmblooded animals which comprises administering to said animals anantibacterially effective amount of antibiotic LL-E19020 Gamma asdefined in claim
 1. 3. A antibiotic pharmaceutical composition whichcomprises an antibiotic amount of LL-E19020 Gamma as defined in claim 1in association with a pharmaceutically acceptable carrier.